There is a continuous need in medical practice, research and diagnostic procedures for rapid, accurate, qualitative or quantitative determinations of biological substances which are present in biological fluids at low concentrations. For example, the presence of drugs, narcotics, hormones, steroids, polypeptides, prostaglandins or infectious organisms in blood, urine, saliva, vaginal secretions, seminal fluids and other biological fluids has to be determined in an accurate and rapid fashion for suitable diagnosis or treatment.
To provide such determinations, various methods have been devised for isolating and identifying biological substances employing specific binding reactions between the substance to be detected (identified as a "ligand" herein) and receptors specifically reactive with that substance. Radioactive or enzyme labels have been used to detect the resulting reactive complex.
One particular type of test which has been developed is what is known in the art as an immunometric or a "sandwich" assay. Such an assay involves "sandwiching" the ligand (such as an antigen) with two or more receptor molecules (such as antibodies) which complex with the compound in a noninterfering manner and at different epitopic sites. Examples of such assays are described in U.S. Pat. No. 4,486,530 (issued Dec. 4, 1984 to David et al) where monoclonal antibodies having high affinity are used. In most sandwich assays, one or more of the receptor molecules are suitably immobilized on an insoluble carrier such as small particles, membranes, plates, or similar objects, as described in U.S. Pat. No. 4,496,654 (issued Jan. 29, 1985 to Katz et al) where a biotinylated antibody is immobilized on an avidin coated support. U.K. Pat. No. 2,074,727 describes sandwich assays in which the complex of antigen and antibodies formed is or will be insolubilized at the time of or subsequent to complex formation.
Avidin is a protein found in egg whites. Biotin, or hexahydro-2-oxo-1H-thieno[3,4]imidazole-4-pentanoic acid (also known as Vitamin H), is a relatively small water-soluble molecule. These materials are known to react specifically with each other to form a very strong and stable complex in which each of the four subunits of avidin binds a biotin molecule. This trong binding is maintained even when either biotin of avidin or both are bound covalently to other materials.
E.P. Publication 201,079 (published Nov. 12, 1986) describes sandwich assays in which the complex of ligand and two receptors is formed in solution. One of the receptors (for example an antibody) is conjugated with either biotin or avidin. A support having biotin or avidin attached thereto is used to insolubilize the resulting ternary complex. While the assay of E.P. 201,079 allegedly provides certain advantages over assays using antibodies directly bound to the insoluble carrier, the assay is carried out by combining individual quantities of all reactants in a test chamber (such as a test tube).
It would be highly useful to have assays designed for mass production in which a minimal number of individually added liquid reagents and mixing steps are needed to determine the ligand. It would be desired to incorporate one or more reagents in a test device used to carry out the assay. U.S. Pat. No. 4,234,316 (issued Nov. 8, 1980 to Hevey) describes a device for delivering measured quantities of reagents into a solution assay medium. The reagents, including antibodies or antigens, are immobilized in the device using any of a wide variety of water-soluble binder materials.
However, there is a further need in the art beyond merely incorporating reagents in a test device for convenient reagent delivery. Those reagents must also be stable for long periods of time. For example, if a kit for an assay is to be transported long distances and stored indefinitely before use, it is necessary that any stored reagents be stable to various environmental conditions during that period of time. Many proteins which might be used as receptor molecules for determination of ligands in patient samples are not generally stable at room temperature for long periods of time. In particular, I have found that the use of biotinylated antibodies or antigens presents a special problem not addressed in the art. For a reason yet unknown, biotinylated antibodies have limited keeping stability.